OBJECTIVES: We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and human gingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types. METHODS: AAV serotypes 1-5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry. RESULTS: Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5. CONCLUSIONS: The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.
OBJECTIVES: We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and humangingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types. METHODS:AAV serotypes 1-5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry. RESULTS: Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5. CONCLUSIONS: The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.
Authors: Sen Yang; Liang Hao; Matthew McConnell; Xuedong Zhou; Min Wang; Yan Zhang; John D Mountz; Michael Reddy; Paul D Eleazer; Yi-Ping Li; Wei Chen Journal: Bone Res Date: 2013-09-01 Impact factor: 13.567